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Polymerization of the backbone of the pectic polysaccharide rhamnogalacturonan I

Nature Plants volume 8, pages 1289–1303 (2022)Cite this article

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An Author Correction to this article was published on 20 March 2023

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Abstract

Rhamnogalacturonan I (RG-I) is a major plant cell wall pectic polysaccharide defined by its repeating disaccharide backbone structure of [4)-α-d-GalA-(1,2)-α-l-Rha-(1,]. A family of RG-I:Rhamnosyltransferases (RRT) has previously been identified, but synthesis of the RG-I backbone has not been demonstrated in vitro because the identity of Rhamnogalacturonan I:Galaturonosyltransferase (RG-I:GalAT) was unknown. Here a putative glycosyltransferase, At1g28240/MUCI70, is shown to be an RG-I:GalAT. The name RGGAT1 is proposed to reflect the catalytic activity of this enzyme. When incubated together with the rhamnosyltransferase RRT4, the combined activities of RGGAT1 and RRT4 result in elongation of RG-I acceptors in vitro into a polymeric product. RGGAT1 is a member of a new GT family categorized as GT116, which does not group into existing GT-A clades and is phylogenetically distinct from the GALACTURONOSYLTRANSFERASE (GAUT) family of GalA transferases that synthesize the backbone of the pectin homogalacturonan. RGGAT1 has a predicted GT-A fold structure but employs a metal-independent catalytic mechanism that is rare among glycosyltransferases with this fold type. The identification of RGGAT1 and the 8-member Arabidopsis GT116 family provides a new avenue for studying the mechanism of RG-I synthesis and the function of RG-I in plants.

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Fig. 1: Representative chemical structure of rhamnogalacturonan I (RG-I) backbone.
Fig. 2: MUCI70 protein domain structure, expression construct and purified protein.
Fig. 3: Recombinant RGGAT1 is an RG-I:Galacturonosyltransferase.
Fig. 4: Biochemical characterization of RG-I:GalAT activity by RGGAT1.
Fig. 5: The combined activities of RGGAT1 and RRT4 polymerize the RG-I backbone.
Fig. 6: Predicted GT-A fold of RGGAT1.
Fig. 7: Phylogenetic tree and tissue expression of GT116 family proteins.

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Data availability

All data generated or analysed during this study are included in this published article (and its supplementary information files) or are available from the corresponding author upon request. UDP-GalA structure was accessed from Protein Data Bank: 3OH1 (https://www.rcsb.org/structure/3OH1). Plant genome sequences were accessed from Phytozome v1357 (https://phytozome-next.jgi.doe.gov/): A. thaliana TAIR10, C. richardii v2.1 (JAIKUY010000000), L. usitatissimum v1.0, M. polymorpha v3.1 (PNPG01000000), O. sativa v7.0, P. virgatum v5.1 (JABWAI010000000), P. trichocarpa v4.1, P. patens v3.3 and S. moellendorffii v1.0. RNA-seq data were accessed from Transcriptome Variation Analysis (http://travadb.org/). Source data are provided with this paper.

Code availability

The code used to predict the glycosyltransferase fold structure was a deep-learning framework previously described in ref. 36, available at https://www.nature.com/articles/s41467-021-25975-9. The published version of the code with the manuscript is available at https://doi.org/10.5281/zenodo.5173136.

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Acknowledgements

Funding was provided by the US Department of Energy, Office of Science, Basic Energy Sciences, Chemical Sciences, Geosciences and Biosciences Division, under award no. DE-SC0015662 (D.M.); the National Institutes of Health Grants P41GM103390 (K.W.M.), R01-GM130915 (K.W.M.) and R35 GM139656 (N.K.); and partially by The Center for Bioenergy Innovation, a US Department of Energy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science (DE-AC05-000R22725, D.M.). We thank M. O’Neill, M. Pena, B. Urbanowicz and P. Prabhakar for technical guidance; S. A. E. Garcia and A. Banks for laboratory support; P. J. Glatz for substrate production; and T. Ishimizu for identifying RRT.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, USA

    Robert A. Amos, Melani A. Atmodjo, Chin Huang, Aarya Venkat, Rahil Taujale, Natarajan Kannan, Kelley W. Moremen & Debra Mohnen

  2. Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA

    Robert A. Amos, Melani A. Atmodjo, Chin Huang, Zhongwei Gao, Kelley W. Moremen & Debra Mohnen

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Contributions

R.A.A. designed and performed experiments. D.M. designed and supervised the research. M.A.A. performed experiments. C.H. and Z.G. completed cell culture and heterologous expression. A.V. and R.T. completed structural prediction and analysis. K.W.M. and N.K. supervised the research. R.A.A. and D.M. wrote the manuscript. All authors reviewed and contributed revisions to the draft.

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Correspondence to Debra Mohnen.

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Extended data

Extended Data Fig. 1 Expression of MUCI70Δ77 in HEK293 cells.

MUCI70Δ77 was expressed in a total of two small-scale (20 mL) and one large-scale (250 mL) cultures. Total protein is the measure of fluorescence of total GFP fluorescence from cells + culture medium. Secreted protein is the measure of fluorescence of cell-free medium. All samples were taken from a 100 µL aliquot from the cell culture after 6 days. MUCI70Δ77 was expressed with 93% secretion efficiency, defined as the proportion of secreted protein to the total protein fluorescence. Error bars represent the standard deviation from three biological replicates.

Extended Data Fig. 2 Digest of RG-I mucilage and purification of RG-I acceptor oligosaccharides.

a, Arabidopsis mucilage was digested for the indicated times with RG-I hydrolase and by acid hydrolysis. Digests were carried out using 10 mg of mucilage and 0.1 µg RG-I hydrolase from Aspergillus aculeatus at 40 °C or 0.1 M HCl at 80 °C for the indicated times. b, RG-I oligosaccharides from digested mucilage were injected into a CarboPac PA-1 semi-preparative (22×250 mm) column following labeling with 2AB. Fractions were collected as individual peaks containing RG-I oligosaccharides of the indicated degree of polymerization (indicated above peak). Peaks were eluted in a gradient ranging from 50-1000 mM ammonium formate indicated by the green line.

Source data

Extended Data Fig. 3 RGGAT1 does not transfer GalA to RG-I acceptors containing GalA on the non-reducing end or to HG acceptors.

a. Hypothetical transfer of GalA to the non-reducing end GalA of an RG-I acceptor, resulting in RG-I oligosaccharides containing at least two contiguous GalA residues on the non-reducing end. Such an enzyme should exist in plants since HG:RG-I heteroglycans are known to be present in plant cell walls. The reaction depicted represents the elongation of homogalacturonan onto an RG-I acceptor. b. RGGAT1 does not catalyze the transfer of GalA to the RG-I (G) acceptor. RGGAT1 (1 mM) was incubated with UDP-GalA and an RG-I (G) acceptor for 1 hour. Longer incubation times did not result in any detectable activity. c. Hypothetical transfer of GalA to the non-reducing end of an HG acceptor, resulting in elongation of the HG backbone by at least one GalA monosaccharide. d. RGGAT1 does not catalyze the transfer of GalA to the HG acceptor. RGGAT1 (1 mM) was incubated with UDP-GalA and an HG acceptor for 1 hour. Longer incubation times did not result in any detectable activity.

Extended Data Fig. 4 Biochemical characterization of RGGAT1 activity.

a, Comparison of RGGAT1 activity using two independent methods. For anion exchange, percentage of acceptor converted was calculated based on the relative proportion of the peaks for the DP12 (R) acceptor and DP13 (G) in the fluorescence chromatogram. For UDP-Glo, activity was measured as a function of UDP released in a 10 min assay containing 1 mM UDP-GalA and 100 µM acceptor. This activity value was presented as “percentage of acceptor converted” based on the conversion that 1 µM UDP released is equal to conversion of 1% of the starting DP12 (R) acceptor to a DP13 (G) product. Reactions contained 50 nM enzyme. Error bars represent the standard deviation from three independent experiments. b, Progress curve of activity using UDP-Glo. In all assays, each point represents the average of duplicate luminescence readings. The blue (assay with 1 mM UDP-GalA) and red (assay with 100 µM UDP-GalA) lines represent the average activity from three independent assays containing 50 nM enzyme. The results from independent assays are shown as individual points. c, Percentage of acceptor conversion was enhanced by addition of a phosphatase (potato apyrase, Sigma A6132) to the reaction. Percentage of acceptor converted was measured as the relative proportion of the peak area of the product to the remaining acceptor at 60 minutes in a reaction containing 50 nM enzyme, 1 mM UDP-GalA, and 100 µM DP12-2AB (R) acceptor. Error bars represent the standard deviation from three independent experiments.

Extended Data Fig. 5 Expression of RRT1, RRT2, RRT3, RRT4, and co-expression of RRT1:RRT2.

Four proteins from the RRT family were expressed in HEK293 cells. A co-expression experiment in which RRT1 and RRT2 were co-transfected into the cells was also performed. Total protein is the measure of fluorescence in the cells + culture medium. Secreted protein is the measure of fluorescence in cell-free medium. Of the four RRT-family proteins expressed in this system, RRT4Δ51 yielded the highest total protein. RRT4 protein expressed with 50% secretion efficiency. Error bars represent the standard deviation of two biological replicates. Co-expression of RRT1Δ61 with RRT2Δ62 did not result in increased expression, suggesting that these two proteins do not form a heterocomplex in vitro.

Source data

Extended Data Fig. 6 The purified RRT4 protein has RG-I:RhaT activity.

The purified RRT4 enzyme was incubated with 1 mM UDP-Rha and an RG-I (G) acceptor, DP12. Activity was tested at pH 6.5 and 7.0 with either 1 µM or 5 µM enzyme. The reaction progress was detected by MALDI-MS at the indicated time points. Activity at pH 6.5 was higher based on the relative conversion of the acceptor (2072 Da) to the RhaT product (2218 Da).

Extended Data Fig. 7 Individual RGGAT1 and RRT4 enzymes do not polymerize RG-I.

RGGAT1 enzyme (1 µM) was incubated with 100 µM RG-I (R) acceptor and 1 mM of UDP-GalA, UDP-Rha, or a combination of UDP-GalA and UDP-Rha. The activity was limited to addition of a single GalA residue with no additional products detected when UDP-Rha was included in the reaction. RRT4 enzyme (1 µM) was incubated with 100 µM RG-I (G) acceptor and 1 mM of UDP-GalA, UDP-Rha, or a combination of UDP-GalA and UDP-Rha. The activity was limited to addition of a single Rha residue with no additional products detected when UDP-GalA was included in the reaction.

Extended Data Fig. 8 Coexpression of RGGAT1 with RRT family members does not improve RRT expression.

RGGAT1 (91.1 kDa) was expressed alone (lane 2) or coexpressed with RRT1 (81.3 kDa), RRT2 (86.4 kDa), RRT3 (86.7 kDa), or RRT4 (85.9 kDa) in HEK293 cells (lanes 3-6). The proteins were purified by Ni2+-NTA affinity from the cell culture medium. Protein concentration was measured by fluorescence. Proteins were loaded into an SDS-PAGE gel based on an equal amount of fluorescence corresponding to an estimated 1 µg total protein. All samples were separated under reducing conditions (+DTT) to observe the presence of monomers. Proteins were compared to previously-purified controls (Lanes 8-10). Lane 10, containing both RGGAT1 and RRT4 protein, was used as a control to demonstrate that the RGGAT1 and RRT4 monomers can be distinguished when an equal amount of both proteins was present. Although some RRT protein may be present in each co-expression lane, the results indicate that they were poorly expressed compared to RGGAT1. The gel represents a single experiment of the coexpression of RGGAT1 with RRT family members.

Source data

Extended Data Fig. 9 DUF616 family sequences are predicted to be a GT-A fold type.

a, Reconstruction error (RE) values are calculated for DUF616 (n = 678) sequences and fall within 95% CI of the RE values for GT-A, B, C and lyso type folds suggesting that DUF616 belongs to one of the known folds. The reference RE values (blue line) were combined from the training set consisting of 39713 GT-A, GT-B, GT-C and GT-lyso sequences. b, RE values for the GT-A (n = 12,316), B (n = 20,397), C (n = 1,518), lyso (n = 5482) and DUF616 (n = 678) sequences are shown as boxplots. Dotted lines mark the 95th and the 99th percentile upper bounds. Boxes show the first and third quartiles. The line within the box indicates the median value. The whiskers mark 1.5 times the interquartile range, excluding the outliers shown as individual diamonds. c, Highest Fold Assignment Scores are found to be for the GT-A1 subcluster for the DUF616 sequences, suggesting that the sequences from this novel family adopt a GT-A type fold. d and e, The RE values against sub cluster GT-A1 and GT-B1 are plotted for DUF616 sequences. As seen, the RE values for GT-A1 are much closer to the true RE values, suggesting overall similarity in core structural fold.

Extended Data Fig. 10 The GT116 family contains eight putative members.

The GT116 domain was annotated as DUF616 (PF04765) in pfam. There are 12 Arabidopsis thaliana DUF616 sequences in pfam corresponding to 8 unique gene loci. The GT116 region is the envelope region containing the DUF616 domain predicted by pfam. The At1g28240/RGGAT1/MUCI70 sequence was entered as a query sequence in Protein BLAST. For the 7 additional GT116 sequences, aligned amino acid residues, query coverage, identity, and similarity are target sequence values obtained using At1g28240 as a query.

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Supplementary Figs. 1 and 2, and Table 1.

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Amos, R.A., Atmodjo, M.A., Huang, C. et al. Polymerization of the backbone of the pectic polysaccharide rhamnogalacturonan I. Nat. Plants 8, 1289–1303 (2022). https://doi.org/10.1038/s41477-022-01270-3

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